Chimeric anti-ricin antibody

ABSTRACT

A chimeric monoclonal antibody targeted to ricin is presented. The light chain and heavy chain constant regions are respectively made up of the light chain and heavy chain constant regions of human immunoglobulin, and the light chain and heavy chain variable regions respectively include the light chain and heavy chain variable regions of macaque immunoglobulin. The antibody does not substantially induce any immune response against chimeric antibodies.

FIELD OF THE INVENTION

This invention concerns a chimeric antibody directed against Ricin.

BACKGROUND OF THE INVENTION

Ricin is a toxalbumin produced by a shrub belonging to the Euphorbiaceaefamily, the Castor Oil Plant (Ricinus communis). Ricin is a highly toxicglycoprotein with a molecular weight of 66 kDa formed by two polypeptidechains A and B connected together by a disulphide bridge. Chain B allowsthe toxin to attach itself to the cell wall while Chain B, which isresponsible for its toxic properties, is capable of inhibiting proteinsynthesis by inhibiting 28S ribosomal RNA, causing cell death. It ispresent in the castor oil seed in concentrations of between 1% and 10%.It may be extracted from incompletely purified castor oil.

As a toxin, ricin is extremely toxic. However, its toxicity variesaccording to the means by which it penetrates the organism.

When ricin is absorbed through digestion, it is largely destroyed byproteolytic digestive enzymes but its perlingual absorption may increasethe quantity absorbed.

In contrast, when ricin is inhaled (pulmonary route) or administered viathe parenteral route its toxicity is multiplied 1000-fold.

Symptoms are fairly non-specific and vary according to the route bywhich the ricin is absorbed. They generally become evident within aperiod of 2 to 24 hours and rarely take longer than 2 days to appear.Absorption through ingestion causes vomiting, feelings of faintness,abdominal pain, bloody diarrhoea (stools resembling rice water), apainful need to defecate or urinate (anuria), dehydration, drowsiness,muscle weakness, cramps, vasomotor paralysis and tachycardia. Absorptionvia inhalation causes weakness, fever, dizziness, dyspnoea, coughing,pulmonary oedemas and pain in the limbs.

After an apparent improvement, infection may have a fatal outcome.

In humans, the dose of ricin estimated to be lethal is between 1 and 10μg/kg.

In view of these varied symptoms and the danger caused by ricin at avery low dose, there is a real need for protection against ricincontamination, including in response to its potential use in the contextof bioterrorist attacks.

A rapid diagnostic test for ricin poisoning via the pulmonary route hasrecently been developed (Guglielmo-Viret et al. 2007). After ricinexposure, the following antidotes may be used: sugar analogues toprevent the ricin from connecting to its target, or catalytic subunitinhibitors such as Azidothymidine.

Vaccination might constitute another ricin poisoning treatment strategy.For example, antibodies have been developed that are designed tointerfer with the connection that the anthrax toxin makes with cellsurface receptors or to inhibit the assembly of the toxin. However, noricin-specific therapy is currently available.

Wang et al. (Wang et al., 2007, Biotechnol Lett 29: 1811-1816) recentlydeveloped a human-mouse chimeric antibody against ricin. However,although this first-generation chimeric antibody is ricin-specific, itis capable of generating a Human Anti-Chimeric Antibody (HACA) immuneresponse and of inducing low patient tolerance.

The international application WO 2009/053637 describes single-chain Fv(scFv) fragments from constant regions of macaque antibodies that arecapable of effectively neutralising ricin, as well as humanized orsuper-humanized scFv fragments.

However, such fragments are small in size, have a very short half-life,are rapidly eliminated by the kidneys and are incapable of providinglong-term protection. Furthermore, given the lack of a constant region,these fragments are relatively ineffective at stimulating the immunesystem (recruitment of immune effectors).

Thus, there is a real need to provide ricin-specific antibodies that arestable after administration and that possess a very high toxinneutralization rate.

It is also important to provide antibodies that do not contribute to aHACA-type immune response in the host when they are administered.

SUMMARY OF THE INVENTION

Thus, the object of the invention is to provide ricin-neutralisingantibodies.

The invention is also intended to provide the means of producing thesericin-specific antibodies.

Another object of the invention concerns the use of these antibodies asmedication or as decontamination agents in response to ricincontamination.

The invention concerns a chimeric monoclonal antibody against ricin, inwhich the light chain and the heavy chain are such that:

-   -   the constant region of the light chain essentially comprises the        constant region of the light chain of a human immunoglobulin;    -   the constant region of the heavy chain essentially comprises the        constant region of the heavy chain of a human immunoglobulin;    -   the variable region of the light chain includes the variable        region of the light chain of a macaque immunoglobulin; and    -   the variable region of the heavy chain includes the variable        region of the heavy chain of a macaque immunoglobulin;        the said monoclonal antibody not substantially inducing a Human        Anti-Chimeric Antibody immune response.

The invention relies on the discovery made by the Inventors thatchimeric macaque/human antibodies under the invention display improvedricin neutralisation, compared to the antibodies or antibody fragmentsused previously, and that the said antibodies are similar to humanantibodies and therefore should not cause a Human Anti-Chimeric Antibodyimmune response.

DETAILED DESCRIPTION OF THE INVENTION

Under the invention, the term “antibody” refers to an immunoglobulin, anoligomeric protein comprising 4 chains that contribute to acquiredimmune response.

Immunoglobulins are familiar to professionals and they consist of acombination of two dimers each comprising a heavy chain and a lightchain. The oligomeric complex is assembled through the connection of alight chain and a heavy chain via a disulphide bridge between twocysteines, the two heavy chains in turn being connected to one anotherby two disulphide bridges.

Each heavy chain and each light chain comprises a constant region and avariable region. The assembly of the constituent chains of an antibodyallow for the definition of a characteristically Y-shapedthree-dimensional structure, where:

-   -   the base of the Y corresponds to the constant region Fc, which        is recognised by the Fc receptors and complement; and    -   the ends of the arms of the Y correspond to the corresponding        assembly of the variable regions of the light chain and of the        heavy chain.        More specifically, each light chain comprises a variable region        (V_(L)) and a constant region (C_(L)). Each heavy chain        comprises a variable region (V_(H)) and a constant region        composed of three constant domains C_(H1), C_(H2) and C_(H3).        The domains C_(H2) and C_(H3) make up the area Fc.

The structure of an antibody is depicted diagrammatically in FIG. 1.

The variable region of the light chain comprises three antigenrecognition determining domains (CDRs) surrounded by four structuraldomains. The three-dimensional folding of the variable region is suchthat the 3 CDRs are exposed on the same side of the protein and allowfor the formation of a special structure to recognise a specificantigen.

The pearl-necklace structure of a variable region of a light or heavychain of an antibody is depicted in FIG. 2.

The antibodies described in the invention are isolated and purified andthey are different from natural antibodies as they are chimeric. Theseantibodies are mature, meaning that they possess a three-dimensional adhoc structure allowing them to recognise the antigen and they possessall the post-translational modifications essential to their antigenrecognition.

They are monoclonal antibodies, meaning that they only recognise asingle antigenic determinant in ricin, unlike polyclonal antibodies,which correspond to a mixture of monoclonal antibodies and can thereforerecognise multiple antigenic determinants within a single protein.

For the purposes of the invention, “chimeric monoclonal antibody” isdefined as an isolated antibody in which the sequence of eachconstituent light chain and/or heavy chain includes or consists of ahybrid sequence derived from at least two different animals. Morespecifically, the chimeric antibodies in the invention are human/macaquehybrids, meaning that a region of the sequence of the light chains andheavy chains derives from the sequence of a macaque immunoglobulin whilethe rest of the sequence of those heavy chains and light chains derivesfrom the sequence of one, or potentially several human immunoglobulins.

“The constant region of the light chain essentially comprises theconstant region of the light chain of a human immunoglobulin” means thatthe constant region of the light chain may comprise the sequence of theconstant region of a human immunoglobulin light chain, but may alsocomprise a sequence corresponding to the fusion of several sequencesfrom several constant regions of several human immunoglobulins. In otherwords, the constant region of the light chain may comprise a sequencecorresponding to a mosaic of sequences from constant regions of lightchains, provided that this mosaic sequence reconstitutes a sequence of aconstant region of a light chain.

“The constant region of the heavy chain essentially comprises theconstant region of the heavy chain of a human immunoglobulin” means thatthe constant region of the heavy chain may comprise the sequence of theconstant region of a human immunoglobulin heavy chain, but may alsocomprise a sequence corresponding to the fusion of several sequencesfrom several constant regions of several human immunoglobulins. In otherwords, the constant region of the heavy chain may comprise a sequencecorresponding to a mosaic of sequences from constant regions of heavychains, provided that this mosaic sequence reconstitutes a sequence of aconstant region of a heavy chain.

“The variable region of the light chain includes the variable region ofthe light chain of a macaque immunoglobulin” means that the sequence ofthe variable region of the light chain corresponds to the sequence ofthe variable region of a macaque immunoglobulin light chain. Thisvariable region of the light chain may be merged into its N-terminalregion, in the C-terminal region of a sequence allowing for theexcretion of the antibody. This sequence allowing for the excretion ofthe antibody is called the signal peptide or leader sequence.

“The variable region of the heavy chain includes the variable region ofthe heavy chain of a macaque immunoglobulin” means that the sequence ofthe variable region of the heavy chain corresponds to the sequence ofthe variable region of a macaque immunoglobulin heavy chain.

This variable region of the heavy chain may be merged into itsN-terminal region, in the C-terminal region of a sequence allowing forthe excretion of the antibody.

Thus, the definition of the monoclonal antibody under the inventioncovers both:

-   -   The precursor of the chimeric human/macaque antibody as defined        above; and    -   The chimeric human/macaque antibody as defined above.

Within the cell that produces the monoclonal antibody covered by theinvention, the said monoclonal antibody against ricin is produced in theform of a precursor. Thus, in the N-terminal region of the light chainand heavy chain, this precursor possesses a leader sequence or signalpeptide. This precursor therefore undergoes various stages ofmaturation, and in particular its leader sequences are cleaved so as toallow the antibody to be secreted in the extra-cellular environment. Thesecreted antibody is therefore a mature antibody.

The monoclonal antibodies under the invention “do not substantiallyinduce a Human Anti-Chimeric Antibody immune response”. This means thatwhen the monoclonal antibodies under the invention are administered toan individual, including a human being, the immune system of the saidindividual is not substantially stimulated or undergoes littlestimulation, and so the said individual does not produce any antibodiesagainst the antibodies covered by the invention. One beneficial methodof producing the invention concerns a monoclonal antibody as definedabove that is capable of neutralising ricin in vitro and in vivo,specifically yielding a ricin neutralisation rate higher than theneutralisation rate of an scFv fragment against ricin.

The Inventors have demonstrated that the antibody according to theinvention is capable of inhibiting ricin more effectively and at a lowerdose than a single-chain fragment of an immunoglobulin (scFv), includinga chimeric human/macaque scFv fragment as defined under the invention

One beneficial method of producing the invention concerns a monoclonalantibody as defined above that is capable of neutralising ricin,specifically yielding a ricin neutralisation rate of at least 40%, 50%,60%, 70% and preferably at least 80%.

The monoclonal antibodies under the invention are “capable ofneutralising ricin”. This means that the monoclonal antibodies accordingto the invention are capable of preventing the action of ricin, or inother words of inhibiting the toxicity of ricin on subunit 28S ofribosomes. This inhibition is due to ricin sequestration or to maskingof the ricin domain or domains responsible for its toxicity.

Consequently, the monoclonal antibodies under the invention neutralisericin toxicity.

The antibody neutralisation activity may be measured with the assistanceof the routine protocol in regular use among professionals. Among thesetests, we can cite the neutralisation test based on cell survival.

This test measures the capacity of the antibodies under the invention toprotect J774A.1 cells put in contact with ricin against death.

In short, J774A.1 cells (ATCC-LGC, Molsheim, France) are cultured at adensity of 14,000 cells/well (200 μl/well) in a culture dish andcultivated at 37° C. with 5% CO₂ for 24 hours in some DMEM to which 10%foetal calf serum is added. The antibodies under the invention areincubated with 480 ng/ml of ricin or with control serum (irrelevantantibodies) for 1 hour. The mixture is then added to the cells. 24 hourslater, cell viability is measured via techniques known to professionals(Trypan blue exclusion, Cytotox (Promega), apoptosis measurement etc).Each test is corrected in relation to cell controls that are “100%viable” (no ricin and no antibodies) and “0% viable” (ricin withoutantibodies).

Another test can be used to measure the neutralising activity of themonoclonal antibodies of the invention, which involves measuring theinhibition of protein translation. For this, one measures thetranslation of a marker protein (e.g. luciferase translation) under anacellular in vitro translation system [Hale M L Pharmacol Toxicol 2001,88(5):255-260].

In short, the luciferase messenger RNA translation assay is as follows:

The monoclonal antibodies are deposited in some Phosphate BufferedSaline (PBS) in 96-well dishes and ricin is added at a finalconcentration of 4 mM. Rabbit reticulocyte lysate complemented withRNAsin®, amino acids and luciferase messenger RNA is then added intoeach well. The reaction lasts for 1 hr 30 mins.

5 μL of the reaction is then sampled and added to 45 μL of reactionbuffer allowing luciferase activity to be detected (Luciferase assayreagent, Promega, Inc.). Light emission (luminescence) is measured incounts per second (CPS) using a Victor multi-plate reader (PerkinElmerWallac, Boston Mass.). The data is expressed as a percentage in relationto the control (% control ═(CPS processed/control CPS)×100)).

Another beneficial method of producing the invention concerns amonoclonal antibody defined above, wherein:

-   -   The constant region of the light chain includes or consists of        the constant region of the light chain of a kappa type human        immunoglobulin; and    -   The constant region of the heavy chain includes or consists of        the constant region of the heavy chain of a type IgG1 human        immunoglobulin;        the said light chain of a kappa type human immunoglobulin and        the said heavy chain of a type IgG1 human immunoglobulin being,        in particular, the constant region of a light chain and the        constant region of a heavy chain of a human immunoglobulin        obtained through immunisation with the Rhesus D antigen.

Thus, the monoclonal antibody under the invention is not only capable ofneutralising ricin via its variable regions, but also of facilitatingricin degradation by promoting its degradation by macrophages.

More specifically, the constant regions of the light chains and heavychains of the monoclonal antibody under the invention derive from theIgG1s of an individual immunised with a Rhesus D antigen. Under anotherbeneficial method of producing the invention, the monoclonal antibodydescribed above and below possesses a light chain and, in particular, aconstant region of the said light chain of the kappa (κ) type.

Under another beneficial method of production, the invention concerns amonoclonal antibody against ricin as described above, wherein:

-   -   The light chain of the said monoclonal antibody includes at        least the complementarity-determining regions (CDR_(Lm)) of the        variable region of a macaque immunoglobulin light chain; and    -   The heavy chain of the said monoclonal antibody includes at        least the complementarity-determining regions (CDR_(Hm)) of the        variable region of a macaque immunoglobulin heavy chain.

The variable region of the light chain of the monoclonal antibody underthe invention therefore includes at least the 3 CDRs of a macaqueimmunoglobulin, while the remainder of the variable region of the lightchain may come from a macaque or from any other mammalian species,including humans.

The variable region of the heavy chain of the monoclonal antibody underthe invention therefore includes at least the 3 CDRs of a macaqueimmunoglobulin, while the remainder of the variable region of the heavychain may come from a macaque or from any other mammalian species,including humans.

When the variable region of the light chain of the monoclonal antibodyonly possesses the CDRs of the light chain of a macaque immunoglobulin,and the variable region of the heavy chain only possesses CDRs from theheavy chain of a macaque immunoglobulin, and all the remainder of thetwo variable regions derives from a human immunoglobulin, the antibodyis then described as a humanized chimeric antibody.

Another beneficial method of producing the invention concerns amonoclonal antibody as defined above, wherein:

-   -   The variable region of the light chain of the said monoclonal        antibody includes or comprises the following SEQ ID No 2        sequence:

(SEQ ID NO 2) Nter-ELQMTQSPSSLSASVGDRVTITCRASQSIRSYLAWYQQKPGKAPKLLIYDAAHLQSGVPSRFSGSGSGTEFSLTISSLQPEDFAVYYCQQRNSYP LTFGGGTKVEIK-Cter;and

-   -   The variable region of the heavy chain of the said monoclonal        antibody includes or comprises the following SEQ ID No 4        sequence:

(SEQ ID NO 4) Nter-QVQLVESGGGLVKPGGSLRLSCAASGFTFTDYYMDWVRQAPGKGLEWVSRISPGGDVTWYADSVKGRFTISRDNAQNTLYLQMNSLRAEDTAVYFCARDDIVVSRIFDDWGQGVLVTVSS-Cter.

Another beneficial method of producing the invention concerns amonoclonal antibody as defined above, wherein:

-   -   The variable region of the light chain of the said monoclonal        antibody comprises the following SEQ ID No 2 sequence:

(SEQ ID NO 2) Nter-ELQMTQSPSSLSASVGDRVTITCRASQSIRSYLAWYQQKPGKAPKLLIYDAAHLQSGVPSRFSGSGSGTEFSLTISSLQPEDFAVYYCQQRNSYP LTFGGGTKVEIK-Cter;and

-   -   The variable region of the heavy chain of the said monoclonal        antibody comprises the following SEQ ID NO 4 sequence:

(SEQ ID NO 4) Nter-QVQLVESGGGLVKPGGSLRLSCAASGFTFTDYYMDWVRQAPGKGLEWVSRISPGGDVTWYADSVKGRFTISRDNAQNTLYLQMNSLRAEDTAVYFCARDDIVVSRIFDDWGQGVLVTVSS-Cter.

REV8

Another beneficial method of producing the invention concerns amonoclonal antibody as defined above, including:

-   -   A light chain including or consisting of the following SEQ ID No        6 sequence:

Nter-ELQMTQSPSSLSASVGDRVTITCRASQSIRSYLAWYQQKPGKAPKLLIYDAAHLQSGVPSRFSGSGSGTEFSLTISSLQPEDFAVYYCQQRNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC-Cter;and

-   -   A heavy chain including or consisting of the following SEQ ID No        8 sequence:

Nter-QVQLVESGGGLVKPGGSLRLSCAASGFTFTDYYMDWVRQAPGKGLEWVSRISPGGDVTWYADSVKGRFTISRDNAQNTLYLQMNSLRAEDTAVYFCARDDIVVSRIFDDWGQGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK-Cter.

REV7

The invention also concerns a monoclonal antibody against ricin, asdefined above, wherein:

-   -   The light chain of the said monoclonal antibody includes the        leader region of the variable region of a human immunoglobulin        light chain (LV_(Lh)),    -   The heavy chain of the said monoclonal antibody includes the        leader region of the variable region of a human immunoglobulin        heavy chain (LV_(Hh)).

Another beneficial method of producing the invention concerns amonoclonal antibody against ricin, as defined above, wherein:

-   -   The N-terminal region of the variable region of the light chain        includes a signal sequence, particularly the leader region of        the variable region of the light chain of a second        immunoglobulin, specifically a human one; and    -   The N-terminal region of the variable region of the heavy chain        includes a signal sequence, particularly the leader region of        the variable region of the heavy chain of a third        immunoglobulin, specifically a human one;        whereby the second and third immunoglobulins may be identical or        different.

As stated above, the leader region or signal peptide, which is locatedin the N-terminal region of the variable region of the light chain andof the heavy chain, corresponds to a protein secretion sequence. Duringprotein synthesis of the light chain and heavy chain, the said leadersequence means that the protein undergoing synthesis must remain in thelight of the Rough Endoplasmic Reticulum (RER). This sequence is theneliminated from the mature light chain and from the mature heavy chain,so that the mature monoclonal antibody capable of interacting with ricinno longer possesses this sequence.

In addition, the invention concerns a precursor of an aforementionedmonoclonal antibody, wherein:

-   -   The leader region LV_(Lh) includes or consists of the following        SEQ ID No 17 sequence:

Nter-MDMRVPAQLLGLLLLWLPGARC-Cter;

-   -   The leader region LV_(Hh) includes or consists of the following        SEQ ID No 18 sequence:

Nter-MKHLWFFLLLVAAPRWVLS- Cter . . . 

Under another preferred means of production, the invention concerns aprecursor of the monoclonal antibody described above, wherein:

-   -   The variable region of the light chain of the said monoclonal        antibody includes or consists of the following SEQ ID No 10        sequence:

Nter-MDMRVPAQLLGLLLLWLPGARCELQMTQSPSSLSASVGDRVTITCRASQSIRSYLAWYQQKPGKAPKLLIYDAAHLQSGVPSRFSGSGSGTEFSLTISSLQPEDFAVYYCQQRNSYPLTFGGGTKVEIK-Cter;and

-   -   The variable region of the heavy chain of the said monoclonal        antibody includes or consists of the following SEQ ID No 12        sequence:

Nter-mkhlwfflllvaaprwvlsQVQLVESGGGLVKPGGSLRLSCAASGFTFTDYYMDWVRQAPGKGLEWVSRISPGGDVTWYADSVKGRFTISRDNAQNTLYLQMNSLRAEDTAVYFCARDDIVVSRIFDDWGQGVLVTVSS-Cter.

In the aforementioned sequences (the sequences SEQ ID No 10 and SEQ IDNo 12) those amino acids underlined whose “single-letter” coding symbolis in lower case correspond to the leader sequence amino acids. Thoseamino acids whose “single-letter” coding symbol is in upper casecorrespond to the variable region.

Under one beneficial method of production, the invention concerns amonoclonal antibody defined above, including:

-   -   A light chain consisting of the following SEQ ID No 14 sequence:

Nter-MDMRVPAQLLGLLLLWLPGARCELQMTQSPSSLSASVGDRVTITCRASQSIRSYLAWYQQKPGKAPKLLIYDAAHLQSGVPSRFSGSGSGTEFSLTISSLQPEDFAVYYCQQRNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC-Cter;and

-   -   A heavy chain consisting of the following SEQ ID No 16 sequence:

Nter-MKHLWFFLLLVAAPRWVLSQVQLVESGGGLVKPGGSLRLSCAASGFTFTDYYMDWVRQAPGKGLEWVSRISPGGDVTWYADSVKGRFTISRDNAQNTLYLQMNSLRAEDTAVYFCARDDIVVSRIFDDWGQGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK-Cter

The invention also concerns the light chain of the monoclonal antibodydefined above, specifically including the sequence SEQ ID No 6, and moreparticularly consisting of the sequence SEQ ID No 6 or SEQ ID No 14.

The invention also concerns the heavy chain defined above, specificallyincluding the sequence SEQ ID No 8, and more particularly consisting ofthe sequence SEQ ID No 8 or SEQ ID No 16.

The invention also concerns a monoclonal antibody fragment as definedabove, the said fragment being a Fab or F(ab)′2 fragment.

scFv fragments are excluded from the invention.

Thus, Fab and F(ab)′2 fragments are constituted of:

-   -   For Fab: a light chain including a constant region of a human        immunoglobulin light chain and a variable region of a macaque        immunoglobulin, and a heavy chain comprising a constant region        of a human immunoglobulin heavy chain and a variable region of a        macaque immunoglobulin;    -   For F(ab)′2, of the combination of the two Fabs described above        via a disulphide bridge.

The object of the invention is also a nucleic acid including a sequencecoding the light chain of the monoclonal antibody defined above, andspecifically including the sequence SEQ ID No 1 or 5

Thus, under the invention the nucleic acid sequences are such that thesequence SEQ ID No 1 codes for the protein SEQ ID No 2, SEQ ID No 3codes for the protein SEQ ID No 4, SEQ ID No 5 codes for the protein SEQID No 6, SEQ ID No 7 codes for the protein SEQ ID No 8, SEQ ID No 9codes for the protein SEQ ID No 10, SEQ ID No 11 codes for the proteinSEQ ID No 12, SEQ ID No 13 codes for the protein SEQ ID No 14 and SEQ IDNo 15 codes for the protein SEQ ID No 16.

The object of the invention is also a nucleic acid including orconstituted of a sequence coding the light chain of the monoclonalantibody defined above, and specifically including the sequence SEQ IDNo 9 or 13.

Another beneficial method of production concerns a nucleic acidincluding or constituted of a sequence coding the heavy chain of themonoclonal antibody as defined above, specifically including thesequence SEQ ID 7.

Another beneficial method of production concerns a nucleic acidincluding or constituted of a sequence coding the heavy chain of themonoclonal antibody as defined above, specifically including thesequence SEQ ID 15.

Another beneficial method of producing the invention concerns a nucleicacid as defined above, including:

-   -   A nucleic acid, coding the light chain of the monoclonal        antibody defined above, including or constituted of a sequence        selected from among the nucleic acids SEQ ID No 1, 5, 9 and 13;        and/or    -   A nucleic acid, coding the heavy chain of the monoclonal        antibody defined above, including or constituted of a sequence        selected from among the nucleic acids SEQ ID No 3, 7, 11 and 15.

Another beneficial method of producing the invention concerns a nucleicacid including or constituted of a sequence coding the light chain ofthe monoclonal antibody as defined above, specifically including orconstituted of any one of the sequences SEQ ID No 1, 5, 9 or 13.

Another beneficial method of producing the invention concerns a nucleicacid including a sequence coding the heavy chain of the monoclonalantibody defined above, specifically including or constituted of any oneof the sequences SEQ ID No 3, 7, 11 or 15.

Another beneficial method of producing the invention concerns a nucleicacid as defined above, including:

-   -   A nucleic acid including or constituted of a sequence selected        from among the nucleic acids SEQ ID No 1, 5, 9 and 13; and    -   A nucleic acid including or constituted of a sequence selected        from among the nucleic acids SEQ ID No 3, 7, 11 and 15.

Another beneficial method of producing the invention concerns a nucleicacid as defined above, including:

-   -   A nucleic acid including or constituted of the sequence SEQ ID        No 5 or 13; and    -   A nucleic acid including or constituted of the sequence SEQ ID        No 7 or 15.

Another beneficial method of producing the invention concerns a nucleicacid as defined above, including:

-   -   A nucleic acid including or constituted of the sequence SEQ ID        No 13; and    -   A nucleic acid including or constituted of the sequence SEQ ID        No 15.

Under another beneficial method of production, the invention concerns anucleic acid including a sequence coding the light chain of themonoclonal antibody defined above and including a sequence coding theheavy chain of the monoclonal antibody defined above.

In other words, the aforementioned sequence includes, within a singlemolecule, or more specifically in the same strand, a sequence coding thelight chain of the monoclonal antibody defined above followed by asequence coding the heavy chain of the monoclonal antibody definedabove. This also means that the aforementioned sequence includes, withina single molecule, a sequence coding the heavy chain of the monoclonalantibody defined above followed by a sequence coding the heavy chain ofthe monoclonal antibody defined above.

The invention also concerns an expression vector including at least onenucleic acid defined above, the said nucleic acid being under thecontrol of elements allowing its expression.

Under the invention, “expression vector” is defined to mean a moleculeof DNA that possesses elements allowing for its replication(duplication) in at least one living organism. In particular, theseelements allowing for replication originate from the replication ofyeast or bacteria, or from elements controlling the replication of avirus.

In particular, according to the invention, the vectors are plasmids,phages, yeast artificial chromosomes (YAC), bacterial artificialchromosomes (BAC), modified genomes of replicative or integrativeviruses etc.

These are known as “expression” vectors because they possess nucleotidesequences that allow expression, namely the transcription into RNA ofthe nucleotide sequences that they control. Under the invention, thesaid nucleic acid sequence contained in the said vector is placed “underthe control of elements allowing its expression”. This means that thesaid expression vector possesses at least one transcription initiationsequence such as a virus promoter like the early promoter of the SimianVirus SV40 or of the Cytomegalovirus (CMV), or sequences promoting RousSarcoma Virus (RSV), and in particular a sequence or promoter includinga TATA box. Furthermore, the said vector also possesses at least onetranscription termination sequence, and in particular a polyadenylationsequence deriving from a mammalian, or more specifically a human gene.

Other sequences allowing for the regulation or modulation of theexpression of the nucleotide sequence contained in the said vector maybe added to those sequences indispensable to the expression of the saidsequence. A non-exhaustive list includes: introns from mammalian, andparticularly human genes, enhancement-type transcription regulationsequences (“enhancers”) or sequences transcribed but not translated frommammalian, and particularly human genes.

One beneficial method of producing the invention concerns an expressionvector as defined above, including at least one nucleic acid selectedfrom among those nucleic acids including the following sequences SEQ IDNo 1, 3, 5, 7, 9, 11, 13 and 15.

Another beneficial method of producing the invention concerns acombination comprising two expression vectors;

the first expression vector including a nucleic acid that includes asequence selected from among the nucleic acids SEQ ID No 1, 5, 9 and 13;andthe second expression vector including a nucleic acid that includes asequence selected from among the nucleic acids SEQ ID No 3, 7, 11 and15.

Another beneficial method of producing the invention concerns acombination comprising two expression vectors, as above, wherein:

The first expression vector includes a nucleic acid that includes thesequence SEQ ID No 13; andThe second expression vector includes a nucleic acid that includes thesequence SEQ ID No 15.

Another beneficial method of producing the invention concerns anexpression vector, as above, including:

-   -   A first nucleic acid selected from among the nucleic acids from        the following sequences: SEQ ID No 1, 5, 9 and 13, the said        first nucleic acid being under the control of elements allowing        its expression; and    -   A second nucleic acid selected from among the nucleic acids from        the following sequences: SEQ ID No 3, 7, 11 and 15, the said        second nucleic acid being under the control of elements allowing        its expression.

Another beneficial method of producing the invention concerns anexpression vector, as above, including:

-   -   A first nucleic acid including or constituted of the sequence        SEQ ID No 13, the said first nucleic acid being under the        control of elements allowing its expression; and    -   A second nucleic acid including or constituted of the sequence        SEQ ID No 15, the said second nucleic acid being under the        control of elements allowing its expression.

This expression vector therefore includes two nucleic acid sequences, asabove, and more specifically it includes a nucleic acid sequence codingthe light chain of the monoclonal antibody defined above, and a nucleicacid sequence coding the heavy chain of the monoclonal antibody definedabove.

By preference, the said expression vector contains a first elementallowing the expression of the nucleic acid sequence coding the lightchain of the monoclonal antibody defined above and a second elementallowing the expression of the nucleic acid sequence coding the heavychain of the monoclonal antibody defined above, the said first and thesaid second element allowing the expression of the said nucleic acidsequences being identical or different, and preferably identical. Inparticular, these control elements are the long terminal repeat (LTR)sequences of the RSV virus.

Another means of producing the invention concerns an expression vectordefined above, including at least one antibiotic resistance gene.

Under the invention, “at least one [ . . . ] resistance gene” is definedto mean that the said expression vector may contain 1 or 2, or 3 or 4 or5 or 6 antibiotic resistance genes.

Under the invention, “antibiotic resistance gene” is defined to mean agene whose expression output exerts a cytostatic (growth inhibiting) orcytolytic (cell death) effect on cells. In particular, the antibioticsconcerned by the invention have an effect on prokaryotic cells, but mayalso have an effect on eukaryotic cells, whether these are yeasts,plants, insects, amphibians or mammals.

More specifically, the aforementioned expression vector possesses anantibiotic resistance gene specific to prokaryotic cells and at leastone or preferably 2 antibiotic resistance genes specific to eukaryoticcells.

The following can be cited as antibiotics specific to prokaryotic cells:Ampicillin, Tetracycline and its derivatives, Hygromycin, Kanamycin etc.The following can be cited as antibiotics specific to eukaryotic cells:G418, Geneticin (G418 salts), Puromycin, Methotrexate, Blasticidin etc.

More particularly, a mode of embodiment of the invention concerns anexpression vector such as previously defined, comprising or consistingof the sequence SEQ ID No 21.

The transcriptional units (TU) of interest coding for the heavy chainand the light chain are cloned under the form of cDNA and under thedependence of the RSV promoter. This promoter corresponds to the LTR(Long Terminal Repeat) of the Rous sarcoma virus which contains anenhancer element in its region 5′.

An artificial intron optimised for alternate splicing is clonedimmediately in region 3′ of the promoter and is composed of a donorsequence in 5′ isolated from the human beta-globin and in region 3′ ofthe acceptor sequence derived from the variable gene of theimmunoglobulin heavy chain. The TU's of interest end by sequences ofpolyadenylation derived from the growth hormone gene (GH) of humanorigin (hGH) for the heavy chain and bovine (bGH) for the light chain.This difference of origin in the choice of polyA is carried out with theaim of limiting the combinations between the genes of interest. Thispromoter association LTRRSV, chimeric intron, cDNA and polyA sequencehas been selected because it confers a high transcriptional andtranslational activity in the cell line YB2/0.

In addition to the UT's of interest the expression vector containsseveral UT's resistant to some chemical molecules:

Bla gene: This gene (named Amp in the vector restriction maps) expressesthe enzyme beta-lactamase in the bacteria (prokaryotic promoter) andconfers a resistance to ampicillin.Neo gene: This gene codes for the nptII enzyme(neomycin-phosphotransferase II) under the control of the SV40 promoterand confers a resistance to various antibiotics such as neomycin,kanamycin or G418 to the transfected mammalian cells expressing thisgene.Dhfr gene: This gene codes for the DHFR enzyme (DiHydroFolate Reductase)under the control of the SV40 promoter and confers a resistance tomethotrexate (MTX). This process can be used to carry out geneamplification by increasing the concentration of MTX resulting from theincrease in production of antibodies by the transfected cells.

The invention also seeks to have a cell, or several cells, or a cellline consisting of at least one expression vector such as previouslydefined.

The cells “consisting of at least one vector “correspond to the cells inwhich at least one expression vector mentioned above has beenintroduced.

Experts in this area of work know perfectly the techniques of molecularbiology which allow the introduction of a DNA sequence or an expressionvector to the interior of a cell, and notably with reference for exampleto (Sambrook, J et al. in Molecular Cloning: A Laboratory Manual. ColdSpring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989). Thereafter theterm “transfection” will be commonly used to describe the action of theintroduction of a vector in a cell.

For example, the techniques of calcium phosphate transfection, by theuse of lipidic particles or “lipofectants”, or by techniques which allowthe generation of holes in the cellular membrane by means of an electricshock (electroporation). This list is not exhaustive.

The cells or cellular lines utilised in the invention are cells fromprokaryotes or eukaryotes such as bacteria, yeast or other mushrooms,insect cells, amphibian cells, mammalian cells and notably rodents,human cells . . . .

The cell is distinguished from the cellular line by the fact that thecellular line is a cellular population in an established culture, thatis to say it has acquired the characteristics which allow theirproliferation in vitro, and notably a characteristic of immortalisation.

A beneficial method of the invention concerns a cell or a cell linepreviously mentioned, presenting a substantially reduced fucosylationactivity compared with a normal cell, the aforementioned cell or cellline being notably a mammalian cell, and in particular the YB2/0 line.

The favourable cell line of the invention is line YB2/0 available at theATCC under number CRL 1662.

In another beneficial method, the invention concerns a cell or a cellline consisting of an expression vector defined above, permitting theexpression of:

-   -   a monoclonal antibody previously defined    -   or of a light chain of a monoclonal antibody such as that        previously described    -   or of a heavy chain of a monoclonal antibody such as that        previously described

In another beneficial method, the invention concerns a cell or a cellline obtained by the cloning of an aforementioned cell.

The techniques of cellular cloning are largely known to experts in thisfield, and are based on the principle of the isolation of cells from acellular population in order that each individual cell generatesdaughter cells (or clones) isolated from the daughter cells from thedivision of other cells of the population.

The general principal of cellular cloning is the limit dilution ofcells.

Also, another beneficial method of the invention concerns a cell or acell line from the aforementioned cloning, the said cells or cell linecharacterised by the fact that they:

-   -   present an apoptosis of below 25%, and    -   secrete at least cloning    -   20 μg/ml of monoclonal antibody previously defined

The measure of apoptosis, or programmed cell death, is made bytechniques routinely employed by experts in this field which involve theevaluation of at least one of the stages characteristic of apoptosis:modification of the plasma membrane, modification of the proteins fromthe Caspase family, modification of the factors of transactions andfragmentation of DNA. Among the cells or cell lines obtained by cloning,the advantage of the invention is that the cells conserved are onlythose which produce a significant quantity of monoclonal antibody, andnotably those which produce at least 20 μg/ml of monoclonal antibody.The measure of the quantity of antibody is easily achievable by theexperts, using simple protein dosage techniques.

Another embodiment of the invention concerns a cell or a cell lineobtained by the cloning of the aforementioned cell, notablycharacterised by the fact that:

-   -   it presents an apoptosis of below 25%    -   it is stable throughout cellular divisions, and    -   it secretes at least 14 μg/ml of monoclonal antibody previously        defined.

The notion of cellular stability implies that the cells from the cloningof cells cloned from cells containing at least one vector permitting theexpression of a monoclonal antibody in accordance with the invention arecapable during the different divisions of conserving their properties ofresistance to antibiotics and of producing the monoclonal antibodies.

A further aspect of the invention concerns the pharmaceuticalcomposition, in particular vaccinal, comprising at least

-   -   one monoclonal antibody defined above, or    -   one nucleic acid defined above, or    -   one expression vector defined above, or    -   one fragment of the said monoclonal antibody defined above,        combined with a vehicle pharmaceutically acceptable.

Advantageously, the invention involves a pharmaceutical composition, inparticular vaccinal, consisting of at least one monoclonal antibodydefined above, combined with an acceptable pharmaceutical vehicle.

The dosage of the active substance depends in particular on the mode ofadministration, and can be easily determined by an expert.

“A pharmaceutically acceptable vehicle” refers to a non-toxic materialwhich is compatible with a biological system such as a cell, a cellularculture, a tissue or an organism.

An effective therapeutic quantity can vary between 0.01 mg/kg and 50mg/kg, preferably between 0.1 mg/kg and 20 mg/kg, and more preferablybetween 0.1 mg/kg and 2 mg/kg, in one or several daily administrations,over one or several days.

The pharmaceutical composition of the product can be administeredintravenously, notably by injection or by gradual drip, subcutaneously,by systemic route, locally by means of infiltration, by bone, or byrespiratory or pulmonary route by means of aerosol.

The preparations for parenteral administration can include sterileaqueous or non-aqueous solutions, suspensions or emulsions. Examples ofnon-aqueous solutions are propylene glycol, polyethylene glycol,vegetable oils, such as olive oil, or injectable organic esters such asethyl oleate. Aqueous vehicles include water, alcohol/water solutions,emulsions or suspensions.

The favourable formulation for the pharmaceutical composition of theinvention is an aerosol comprising

-   -   a monoclonal antibody defined above, or    -   a nucleic acid defined above, or    -   an expression vector defined above, or    -   a fragment of the said monoclonal antibody defined above,        combined with an excipient, with or without a propulsion agent.

In one embodiment of the product, the aerosol presents in the form of aliquid containing the anti-ricin antibody and an excipient. Usually theexcipient is alcohol, but any other excipient known by the experts couldbe utilised within the framework of the invention. A liquid form aerosolcan be linked to a gas propellant such as chlorofluorocarbon (CFC) orhydro fluorocarbon (HFA).

The aerosol in liquid form can also be made up of lipidic microparticles and an excipient. In this case the excipients can be chosenfrom synthetic dipalmitoylphosphatidylcholine (DPPC), lactose orhydroxyethylamidon (HES). The micro particles are then administered withthe aid of an insufflator.

In another embodiment of the invention the aerosol presents in powderform. The powder is composed of particles of sizes of between 1 and 10μm and preferably smaller than 9 μm, or smaller than 5 μm. As anon-exhaustive guide, the following methods can be used to obtain a drypowder: pulverisation together with desiccation by freezing orcrystallisation by ultrasound, controlled precipitation.

The administration of the aerosol will be carried out according towhether it presents in liquid or solid form with the aid of a nebuliserwhich can be pneumatic, ultrasonic or sieved or with the aid of ametered dose inhaler (pressurised liquid, mechanic, electrodynamic,thermic) for liquid formulations or with the aid of an inhaler for solidformulations. (Reychler G., Dessanges J F and Vecellio L, RespiratoryJournal, 2007; 24:1013-1023).

The invention also includes the utilisation of at least:

-   -   one monoclonal antibody defined above, or    -   one fragment of the said monoclonal antibody defined above,    -   one nucleic acid defined above, or    -   one expression vector defined above, or    -   one cell defined above,        for the preparation of a drug for the treatment or prevention of        a pathology linked to ricin contamination.

By treatment, we mean the method of treatment for a manifestedpathology, whose symptoms are visible. By prevention, we mean the methodof stopping the said pathology from manifesting.

Pathologies associated with ricin correspond to symptoms linked to ricincontamination, in particular diarrhoea, changes in electrolytes,dehydration, swelling, and respiratory, hepatic and renal problems . . ..

Equally the invention aims to achieve in vitro dosage of ricin in abiological sample taken from an individual susceptible to ricincontamination, the aforementioned method comprising:

-   -   placing the said sample in contact with at least one monoclonal        antibody as previously defined, and    -   the determination of the presence or absence of ricin in the        said sample by the detection of the formulation of an eventual        immune complex between ricin and the said monoclonal antibody.

The monoclonal antibodies of the invention can therefore be utilised todetect the presence of ricin in a biological sample.

In particular, the said antibodies can be utilised for theimplementation of detection techniques known by experts such as ELISA,RIE, immunoprecipitation or immunolabelling. (Western blot).

The invention also concerns an in vitro decontamination procedure, for asample vulnerable to contamination by ricin, particularly a biologicalsample, which involves:

-   -   the placement of the said sample in contact with at least one of        the above monoclonal antibodies, and    -   the elimination from the said sample of the immune complexes        formed between ricin and the said monoclonal antibody.

A means of implementing the said decontamination procedure consists, forexample, in the use of the invention's monoclonal antibodies, upon whichmagnetic spheres are grafted to the constant region of the saidantibody.

Once the aforementioned antibodies, coupled with magnetic spheres, areplaced in contact with the sample to be decontaminated, the ricin iseliminated from the sample by capturing the antibodies according to theinvention, with magnets.

Another means to implement the said decontamination procedure consists,for example, in the use of monoclonal antibodies that have beenimmobilised on a column on which Staphylococcus aureus protein A or G ispresent. The sample to decontaminate is then passed through the columnin order to capture, on the antibodies, according to the immobilisedinvention, any ricin which may be contained in the said sample.

One beneficial method of producing the invention concerns a procedurefor the preparation of an aforementioned anti-ricin monoclonal antibody,which involves:

-   -   a. the transformation of a cell, particularly that of a mammal,        specifically the YB2/0 line, with at least one vector defined        above;    -   b. the selection of the transformed cells;    -   c. the evaluation of the production of the said antibody by the        clones selected in the preceding stage, by determining the        presence or absence of the formation of any immune complexes        between the ricin and the said monoclonal antibody.

One beneficial method of producing the invention concerns a proceduredefined above, in which the selection of transformed cells is achievedby determining their resistance to at least one antibiotic.

One beneficial method of producing the invention concerns a proceduredefined above, in which the cells producing a quantity of the saidmonoclonal antibody greater than 22 μg/mL are selected.

One beneficial method of producing the invention concerns a proceduredefined above, in which the cells selected in Step C are cloned.

One beneficial method of producing the invention concerns a proceduredefined above, in which the cells selected in Step C are re-cloned for:

-   -   their resistance to at least one antibiotic, and    -   their capacity to produce at least 11 μg/mL of the said        monoclonal antibody.

The invention is better illustrated by the following examples andfigures. The examples below aim to clarify the object of the invention,and to illustrate beneficial methods of realising it, but in no waylimit the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 corresponds to a schematic representation of an antibody. Theblack parts correspond to the constant regions of the heavy chains, thedark grey parts correspond to the constant region of the light chain,the parts in light grey correspond to the variable region of the heavychain, and the white parts correspond to the variable region of thelight chain. —S—S— represents the disulphide bridges between twocysteines. The CDR regions and structures are indicated by arrows. TheFab and Fc fragments are also represented.

FIG. 2 corresponds to a schematic pearl-necklace representation of theamino acid sequence of a variable section of the light chain or heavychain of immunoglobulin. The black circles correspond to the amino acidsforming the structured regions, and the grey circles correspond to theamino acids representing the CDRs.

FIG. 3 corresponds to a schematic representation of the bond between theleader peptide of the variable section of the heavy chain of a humanimmunoglobulin and the variable section of the heavy chain of a macaqueimmunoglobulin. The oligonucleotides which have served as PCRs are drawnon the schematic. The unique restriction sites are also indicated.

FIG. 4 corresponds to a schematic representation of the bond between theleader peptide of the variable region of the heavy chain of a humanimmunoglobulin and the variable region of the light chain of macaqueimmunoglobulin. The oligonucleotides which have served as PCRs are drawnon the schematic. The unique restriction sites are also indicated.

FIG. 5 corresponds to the schematic representation of the intermediatecloning vector H622-26 containing the ‘double hybrid’ heavy chain, inwhich the human leader is bound to the variable region of the macaqueheavy chain (VH 43RCA), which is itself bound to the constant region ofthe human immunoglobulin (CH T125).

The different regulation elements (promoters, chimeric introns,polyadenylation sites, etc.), as well as the genes for resistance toantibiotics and the origins of replication, are also represented.

FIG. 6 corresponds to the schematic representation of the final cloningvector HK622-26 (SEQ ID No. 21) containing the ‘double hybrid’ heavychain in which the human leader is bound to the variable region of themacaque heavy chain (VH 43RCA), which is itself bound to the constantregion of the human immunoglobulin (CH T125), and the ‘double hybrid’light chain in which the human leader is bound to the variable region ofthe macaque light chain (VK 43RCA), which itself is bound to theconstant region of the human immunoglobulin (CK T125).

The different regulation elements (promoters, chimeric introns,polyadenylation sites, etc.), as well as the genes for resistance toantibiotics and the origins of replication, are also represented.

FIG. 7 corresponds to a 2% agarose gel on which the PCR productsallowing the detection of the mycoplasms have been separated.

Bands 3 and 22 correspond to the migration of the molecular weightmarker; bands 1 and 20 correspond to the migration of the PCR productsmade from a negative control, and bands 2 and 21 correspond to themigration of PCR products made from a positive control.

Bands 4, 6, 8, 10, 12, 14, 16, 18, 23, and 25 correspond to themigration of the PCR products made respectively from the cloid basesGG3, IF2, EE9, EG11, JB3, ED9, EC2, GF2, 1A1 and KC9.

Bands 5, 7, 9, 11, 13, 15, 17, 19, 24, and 26 correspond to themigration of the PCR products made respectively from the cloid basesGG3, IF2, EE9, EG11, JB3, ED9, EC2, GF2, 1A1 and KC9.

FIG. 8 represents in graph form the stability of antibody production forthe three cloids EE9, GG3 and IF2. The grey bars represent measurementstaken during the first dosage, and the black bars represent measurementstaken during the second dosage.

The abscissa or x-axis represents time, in days, and the ordinate ory-axis represents the quantity of antibodies produced in ng/mL.

FIG. 9 corresponds to a 2% agarose gel on which the PCR productsallowing the detection of the mycoplasms have been separated.

Bands 4, 17, 30 and 33 correspond to the migration of the molecularweight markers; bands 3 and 31 correspond to the migration of the PCRproducts made from a negative control, and bands 2 and 32 correspond tothe migration of PCR products made from a positive control. Bands 5, 7,9, 11, 13, 15, 18, 20, 22, 24, 26 and 28 correspond to the migration ofthe PCR products made respectively from the clone bases GG9-4B2,GG9-2F8, GG9-5D8, GG9-2G10, IF2-2F2, IF2-2E3, GG3-2G4, GG3-EC2, GG3-1G9,GG3-2G2, GG3-5G11, GG3-1D8 and EE9-5G7.

Bands 6, 8, 10, 12, 14, 16, 19, 21, 23, 25, 27 and 29 correspond to themigration of the PCR products made respectively from the clone basesGG9-4B2, GG9-2F8, GG9-5D8, GG9-2G10, IF2-2F2, IF2-2E3, GG3-2G4, GG3-EC2,GG3-1G9, GG3-2G2, GG3-5G11, GG3-1D8 and EE9-5G7.

FIG. 10 represents in graph form the stability of antibody productionfor the clones EE9-2G10, EE9-5D8, EE9-5G7, GG3-1G9, GG3-2G4, IF2-1C7,IF2-2D8 and IF2-2E9 over 8 days. The grey bars represent measurementstaken during the first dosage, and the black bars represent measurementstaken during the second dosage.

The abscissa or x-axis represents time, in days, and the ordinate ory-axis represents the quantity of antibodies produced in ng/mL.

FIG. 11 represents the cumulative Kaplan-Meier curve for mice havingreceived 50 μg of ricin by pulmonary instillation as well as a controlhuman IgG (curve with triangles), or the antibody according to theinvention (43RCA), 6 hours (curve with squares), 22 hours (curve withdiamonds) or 24 hours (dashed curve with circles) after the instillationof the ricin.

FIG. 12 represents the cumulative Kaplan-Meier survival curve for micehaving received 50 μg of ricin by pulmonary instillation as well as 20μg of control human IgG (curve with triangles), or the antibodyaccording to the invention (43RCA) at a concentration of 20, 10, 5 and 1μg;

FIG. 13 represents the cumulative Kaplan-Meier survival curve for micehaving received 50 μg of ricin by pulmonary instillation as well as 150μg of the antibody according to the invention (43RCA), 44 hours (curvewith squares), 54 hours (curve with diamonds) after the instillation ofthe ricin. A control (the curve with triangles) indicates the survivalrate of mice treated with the ricin and a non-relevant immunoglobulin.

EXAMPLES Example 1 Construction and Sequencing of an HK622-26 ExpressionVector for the Expression of Antibodies According to the Invention

The expression vector HK622-26 (SEQ ID No 21) was constructed for theexpression of the chimeric macaque-human (IgG) anti-ricin monoclonalantibody.

The HK622-26 vector was constructed from the CHK622-05 vector by ‘doublechimerization,’ meaning the addition by PCR of assemblies from humanleader regions, and by a cloning addition of the constant CK and CHhuman regions to the VH and VK macaque variable sequences.

The variable heavy and light chain VH and VK regions are extracted froma coding vector for an anti-ricin ScFv, ScFv43RCA/H2-V116, and areintroduced into the ‘generic’ CHK622-08 vector, after adding leadersequences to ensure a good synthesis of the chimerical antibody. The CKand CH constant regions are of human origin, and are derived from cloneT125-A2, directed against the Rhesus D antigen.

A—Synthesis of the VH43RCA Region by PCR Assembly

The VH43RCA region corresponds to the chimerization of:

-   -   the leader VH sequence of the human M29812 sequence (in groups        VH4, VH4-59), contained in the vector HK588-12, where the said        leader VH sequence has been constituted in the following manner:

(SEQ ID NO 19) 5′-ATGAAACATCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCC-3′,and

-   -   the macaque VH anti-ricin region contained in the plasmid phAi14        containing the fragment ScFv43RCA/H2-V116, where the said VH        macaque sequence has been constituted from the SEQ ID No 3        sequence.

The chimerization is performed in the following manner:

-   -   the leader VH sequence (SEQ ID No 19) is amplified by PCR using        the following initiator pair:

VH1-Ricin sense initiator: (SEQ ID NO 22) 5′-CTCAGTGCTAGCGCCGCCACCATGAAACATCTGTGGT-3′ VH2-Ricin antisense initiator:(SEQ ID NO 23) 5′-CCAGCTGCACCTGGGACAGGACCCAT-3′starting from a plasmidic HK558-12 DNA matrix.

The PCR reaction is performed according to the following protocol:

-   -   denaturation: 5 min. at 95° C.    -   denaturation: 0.5 min. at 95° C.┐    -   hybridisation: 0.5 min at 50° C.} 10 repetitions    -   elongation: 0.5 min. at 72° C.┘    -   elongation: 10 min. at 72° C.

This pair of initiators allows the creation of an amplicon (amplicon 1)of 91 bases containing the site Nhe I (GCTAGC), the Kozak sequence(bold) and the human leader sequence SEQ ID No 17).

-   -   the macaque VH sequence (SEQ ID No 3) is amplified by PCR using        the following initiator pair:

VH3-Ricin sense initiator: (SEQ ID NO 24)5′-ATGGGTCCTGTCCCAGGTGCAGCTGG-3′ VH4-Ricin antisense initiator:(SEQ ID NO 25) 5′-ACCGAT GGGCCC TTGGTGGAGGCTGAGGAGACGGTGACCA-3′,starting with a plasmidic phAi14 DNA matrix.

The PCR reaction is performed according to the following protocol:

-   -   denaturation: 5 min. at 95° C.    -   denaturation: 0.5 min. at 95° C.┐    -   hybridisation: 0.5 min at 50° C.} 10 repetitions    -   elongation: 0.5 min. at 72° C.┘    -   elongation: 10 min. at 72° C.

This initiator pair allows the creation of an amplicon (amplicon 2) of396 bases containing the macaque VH sequence and the site Apa I(GGGCCCC).

Amplicons 1 and 2, previously obtained, are then combined to give thefinal amplicon 3 by PCR assembly using the aforementioned VH1 and VH4initiators.

The PCR reaction is performed according to the following protocol:

-   -   denaturation: 5 min. at 95° C.    -   denaturation: 0.5 min. at 95° C.┐    -   hybridisation: 0.5 min at 50° C.} 15 repetitions    -   elongation: 1 min. at 72° C.┘    -   elongation: 10 min. at 72° C.

FIG. 3 shows the VH43RCA region with the different initiator pairshaving served in the preparation of the final chimerical VH fragment(amplicon 3) of 461 bp.

B-Synthesis of the VK43RCA Region by PCR Assembly

The VK43RCA region corresponds to the chimerization of:

-   -   the leader VK sequence of the human Z0006 sequence (in groups        VK1, VK1-13), contained in the vector HK588-12, where the said        leader VK sequence has been constituted in the following manner:

(SEQ ID NO 17) 5′-ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGATGT-3′,and

-   -   the macaque VK anti-ricin region contained in the plasmid phAi14        containing the fragment ScFv43RCA/H2-V116, where the said VH        macaque sequence has been constituted from the SEQ ID No 1        sequence.

The chimerization is performed in the following manner:

-   -   the leader VK sequence (SEQ ID No 17) is amplified by PCR using        the following initiator pair:

VK1-Ricin sense initiator: (SEQ ID NO 26) 5′-CTCAGTACTAGTGCCGCCACCATGGACATGAGGGTCCCCG-3′ VK2-Ricin antisense initiator:(SEQ ID NO 27) 5′-TGTCATCTGGAGCTCACATCTGGCACCTGG-3′starting from a plasmidic HK558-12 DNA matrix.

The PCR reaction is performed according to the following protocol:

-   -   denaturation: 5 min. at 95° C.    -   denaturation: 0.5 min. at 95° C.┐    -   hybridisation: 0.5 min at 50° C.} 10 repetitions    -   elongation: 0.5 min. at 72° C.┘    -   elongation: 10 min. at 72° C.

This pair of initiators allows the creation of an amplicon (amplicon 1′)of 102 bases containing the site Spe I (ACTAGT), the Kozak sequence(bold) and the human leader sequence (SEQ ID No 19).

-   -   the macaque VK sequence (SEQ ID No 1) is amplified by PCR using        the following initiator pair:

VK3-Ricin sense initiator: (SEQ ID NO 28)5′-CCAGGTGCCAGATGTGAGCTCCAGATGACA-3′ VK4-Ricin antisense initiator:(SEQ ID NO 29) 5′-TGAAGA CACTTGGTG CAGCCACAGTTCGTTTGATCTCCACCTTGGTC C-3′starting from a plasmidic phAi14 DNA matrix.

The PCR reaction is performed according to the following protocol:

-   -   denaturation: 5 min. at 95° C.    -   denaturation: 0.5 min. at 95° C.┐    -   hybridisation: 0.5 min at 50° C.} 15 repetitions    -   elongation: 1 min. at 72° C.┘    -   elongation: 10 min. at 72° C.

This initiator pair allows the creation of an amplicon (amplicon 2′) of364 bases containing the macaque VK sequence and the Dra III site(CACTTGGTG).

Amplicons 1′ and 2′, previously obtained, are then combined to give thefinal amplicon 3′ by PCR assembly using the aforementioned VK1 and VK4initiators.

The PCR reaction is performed according to the following protocol:

-   -   denaturation: 5 min. at 95° C.    -   denaturation: 0.5 min. at 95° C.┐    -   hybridisation: 0.5 min at 50° C.} 15 repetitions    -   elongation: 1 min. at 72° C.┘    -   elongation: 10 min. at 72° C.

FIG. 4 shows the VK43RCA region with the different initiator pairshaving served in the preparation of the final chimeric VH fragment(amplicon 3′) of 436 bp.

C-Construction of Vector H622-26

Amplicons 3 and 3′ are introduced into the ‘generic’ vector CHK622-08containing the constant CH heavy and CK light chain sequences from theIgG1 of the clone T125-A2 directed against the Rhesus D antigen.

The cloning of amplicons 3 and 3′ is performed sequentially in thefollowing way:

1—Amplicon 3 and the vector CHK622-08 are subjected to a doubledigestion by the restriction enzymes Nhe I−Apa I.

The digestion fragments are then purified, and a vector/amplicon 3mixture is subjected to a cohesive ligation.

The products of the ligation are then used to transform bacteria, andthe transformers are selected on an LB medium with an ampicillincomplement.

The resistant clones are then tested by PCR by using the initiatorsSPRSVBIS and GSP2ANP: a fragment of 783 bp is expected for the positiveclones.

6 positive clones in PCR were selected for a screening via enzymaticdigestion:

-   -   by Nhe I+Apa I control digestion (verification of the insert and        junctions): The expected profile consists of 2 strips, 447+9917        bp, and    -   by Hind III digestion (verification of the entire vector): The        expected profile consists of 4 strips, 3759+2894+2559+1152 bp.

All of the clones are positive, and the H622-26 clone, whose restrictionmap is represented in FIG. 5, is chosen for the second step.

2—Amplicon 3′ and the vector H622-26 are subjected to a double digestionby the restriction enzymes Spe I−Dra III.

The digestion fragments are then purified, and a vector/amplicon 3′mixture is subjected to a cohesive ligation.

The products of the ligation are then used to transform bacteria, andthe transformers are selected on an LB medium with an ampicillincomplement.

The resistant clones are then tested by PCR by using the initiators5′1PLC and CK4: a fragment of 539 bp is expected for the positiveclones.

6 positive clones in PCR were selected for a screening via enzymaticdigestion:

-   -   by Spe I+Dra III control digestion (verification of the insert        and junctions): The expected profile consists of 2 strips,        420+10344 bp, and    -   by Hind III digestion (verification of the entire vector): The        expected profile consists of 4 strips, 3759+2894+2559+1151 bp.

All of the clones are positive. After sequencing, only 4 clones possessa correct sequence. Clone 2 was chosen, and corresponds to vectorH622-26. The restriction map of the vector H622-26 is represented inFIG. 6.

The vector is thus ready for the transformation of cells.

Example 2 Obtaining Anti-Ricin-Producing Clones in the YB2/0 Line byDirect Double Transfection

This study has the aim of obtaining clones producing anti-ricin in theYB2/0 line by direct double transfection.

The YB2/0 cells were maintained (by re-treatment with 1×10⁵ cell/mltwice per week) in light of transfection at 10⁵ cells/ml in an EMSmedium, 5% SVF.

The vectors utilised for the transfection are as follows:HK622-26/EcoRV, H416-24 (T+) et K416-23 (T+). HK622-26/EcoRV signifiesthat the vector obtained in example 1 was linearised by digestion withthe Eco RV enzyme in order to promote its integration into thetransformed cells.

Transfection

The cells are transfected according to the following protocol:

4 cuvettes containing 500 μL of cells are prepared in the followingmanner:

-   -   Cuvettes 1 and 2: 42.8 μg of vector HK622-26/EcoRV    -   Positive control cuvette: 25.2 μg of vector H416-24, linearised;        -   23.2 μg of vector H416-23, linearised;    -   Negative control cuvette: no vectors.

The cells are then subjected to electrophoresis at a voltage of 230 Vand a capacitance of 960 μF for 17.9 ms.

Once the electrophoresis is complete, the cells are carefully removedfrom the cuvettes and placed in a selective medium, RPMI, 5% dialysedSVF, 1 g/L geneticin for the P24 (25,000 cells/ml) and P96 at 100cells/well (geneticin being applied only at J+3, once the resistancegene has been expressed) and in RPMI, 5% dialysed SVF, 0.5 g/Lgeneticin, 50 nM methotrexate (MTX) and seeded in culturing dishes inthe following manner:

-   -   Cuvettes 1 and 2 (per cuvette): 1 plate, 24 wells, at 25,000        cells/well        -   5 plates, 96 wells, at 2500 cells/well        -   1 plate, 96 wells, at 100 cells/well    -   T+Control cuvette: 1 plate, 96 wells, at 2500 cells/well        -   1 plate, 96 wells, at 100 cells/well    -   T−Control cuvette: 1 plate, 96 wells, at 2500 cells/well        -   1 plate, 96 wells, at 100 cells/well

The cells are then cultivated in the presence of antibiotic for 4 weeks.

For the 96 well plates, the medium is changed twice per week.

The rest of the transformed cells are spread over 6 wells for each ofcuvettes 1 and 2.

Selection of Transformers

Evaluation of the Production of Antibodies

In an initial step, the mean rate of antibody production is evaluatedover 3 pools from each of the transfections of cuvettes 1 and 2, after13 days in a selective culture medium.

In total 6 pools were tested, and the mean concentration of monoclonalantibodies was measured in μg/mL. The cellular density and cellularviability were also determined.

The results obtained are presented in Table 1 below:

TABLE 1 Viability and dosage of antibody production from the variouspools. Antibody Cellular Density Viability Concentration Cuvette Pool(10⁶ cells/ml) (%) (μg/ml) 1 1 0.62 82 5.6 2 0.52 74 5.1 3 0.44 88 4.2 21 0.62 84 3.6 2 0.72 86 5.8 3 0.52 72 6

We observe that the two transfections allow the production of anti-ricinmonoclonal antibodies.

First Clonal Selection

After 22 days of culture in a selective medium, the appearance of clonesresistant to G418 and to methotrexate was evaluated.

The results of the clones appearing are described in Table 2 below:

TABLE 2 Number of clones having emerged. Number of Number of wells wellsNumber of where where cells clones clones Cuvette seeded Number emergedemerged/ No. Selection per well of P96 (total) P96 1 RPMI + 5% SVF 25005 34 6.8 dialysed + G418 0.5 g/L + 25 nM MTX 2 RPMI + 5% SVF 2500 5 428.4 dialysed + G418 0.5 g/L + 25 nM MTX T+ RPMI + 5% SVF 2500 1 1 1dialysed + G418 0.5 g/L + 25 nM MTXP96=96-well plates

In total 76 double resistant (G418 and methotrexate) pseudo-clones(wells) emerged following the double selection, for a transfectionefficiency of 7.9% (76 positive wells for 960 seeded wells).

The cells which had been grown in the wells are considered as not beingpure clones, as 2500 cells per well were seeded.

For each of the pseudo clones, or cloids, the quantity of immunoglobulinsecreted was measured.

The results of the measurements from the best 24 clones are shown inTable 3 below:

TABLE 3 quantities of antibodies produced for the different cloidstested. Name Average of the of the 1 IgG ng/ml 2 IgG ng/mL 3 IgG ng/mL 3IgG ng/mL cloid quantity quantity quantity quantities measured BA1 48005862 5843 5502 CB8 1700 1978 2253 1977 CE10 1100 1283 1413 1265 DH6 7001405 2809 1638 EC2 12300 13153 21402 15618 EC9 9300 10638 11428 10455ED9 12300 13734 15564 13866 EE9 22000 21738 24161 22633 EG11 16800 1571618330 16949 GF11 6100 5529 6125 5918 GF2 11000 11715 13187 11967 GG327000 27371 28737 27703 HC9 5200 6386 7151 6246 HE7 8000 11015 1303610684 HE8 1400 1492 1764 1552 IA1 10000 11779 12322 11367 IA6 6300 49787044 6107 IA7 3800 4887 5493 4727 IF2 26000 29707 31385 29031 JB3 1650014736 17530 16255 JB9 6600 6973 7970 7181 JE6 8200 8626 9557 8794 KB6400 899 1165 821 KC9 9900 7996 8832 8909

Of the 24 cloids tested, 10 had a monoclonal antibody production of morethan 8.9 μg/mL. All these cloids (EC2, ED9, EE9, EG11, GF2, CG3, IA1,IF2, JB3 and KC9) were deep frozen in a medium without antibiotic, inthe presence of 10% DMSO, and kept at −195° C. in liquid nitrogen.

A cell viability test was conducted for each of these 10 cloids beforefreezing (Table 4) and they were screened for the possible presence ofmycoplasma (FIG. 7).

All the clones had a satisfactory survival rate (>85%) and no clone wascontaminated by mycoplasma.

TABLE 4 viability of the cloids Cloid Viability (%) GF2 89 EE9 97 IF2 89EG11 89 KC9 85 EC2 95 ED9 95 GG3 100 IA1 100 JB3 100

The 3 cloids (EE9, CG3 and IF2) whose production of antibodies washighest (more than 22 μg/mL) were selected for further cell cloning.

The stability of the monoclonal antibody production was also tested foreach of the cloids selected.

The three cloids EE9, GG3 and IF2 were kept for 6 weeks by cascadedilution in 24 well plates in a DMEM medium+5% dialysed SVF andsubcultured every 7 days. The production of monoclonal antibodies wasevaluated in two independent ELISA assays and the results are shown inTable 5 below:

TABLE 5 antibody production over time. Name Name Name of the QuantityQuantity of the Quantity Quantity of the Quantity Quantity cloid Week 1(ng/ml) 2 ng/mL cloid Week 1 (ng/ml) 2 ng/mL cloid Week 1 (ng/ml) 2ng/mL EE9 1 13800 18415 GG3 1 14600 19131 IF2 1 12700 19743.5 EE9 217000 22901.5 GG3 2 12500 18022 IF2 2 16300 21009.5 EE9 3 17500 25188.5GG3 3 9500 13153.5 IF2 3 12800 15733.5 EE9 4 14400 23695 GG3 4 72009257.5 IF2 4 7800 12186.5 EE9 5 16100 23878.5 GG3 5 5800 7270.5 IF2 510700 11822.5 EE9 6 15000 19890.5 GG3 6 3000 4433.5 IF2 6 7100 10581.5

The stability of the cloids over time is also represented in graphicform in FIG. 8.

Of the 3 cloids selected, the GG3 cloid saw its production of antibodiesgo down very significantly during the various stages.

On the basis of this information, the cloids selected were recloned inorder to achieve “pure” clones.

Second Clonal Selection

A second cloning of the cloids EE9, CG3 and IF2 was conducted in DMEM 5%dialysed SVF medium. 5×96 well plates, with 40 cells per well, wereseeded in a DMEM 5% dialysed SVF medium. There was no clone for thiscloning.

A second cloning was carried out in a DMEM and EMS 5% dialysed SVFmedium.

The cells were unfrozen in a RPMI 5% dialysed SVF medium then passedinto an EMS 5% dialysed SVF and DMEM 5% SVF medium. The cells weresubcultured to 4.5.10⁵ cellules/mL on the eve of cloning.

5×96 well plates, with 40 cells per well, were seeded per cloid and permedium, namely a total of 30 plates, or 2880 wells. The numbers ofclones which appeared are shown in Table 6.

TABLE 6 Table 6: number of clones which emerged after cloning the 3cloids, as a function of the culture medium. Number of clones appearingMedium Cloids DMEM EMS IF2 22 1 GG3 30 0 EE9 2 25

Two siftings using ELISA allowed the 10 best clones to be selected fromthe 80 cloid clones which emerged.

The ELISA results from the 11 clones produced from cloid EE9, the 11clones produced from cloid GG3 and the 10 clones produced from cloidIF2, are shown in Table 7 below:

TABLE 7 quantities of antibodies produced for the different cloidstested. Quantity 1 Quantity 2 Average E-IgG Culture Stage ng/mL ng/mLng/mL EE9-5G7 17 536  21 002  19 269  EE9-5D8 14 572  14 647  14 610 EE9-2G10 14 901  15 642  15 272  EE9-2F8  4 198 5 433 4 816 EE9-4B1  2637 6 475 4 556 EE9-1B6  3 773 4 865 4 319 EE9-1G5  3 571 2 807 3 189EE9-3G4  1 715 2 255 1 985 EE9-3G2  793 1 544 1 543 EE9-4H3 <1600 1 0311 031 EE9-4G9 <1600   910   910 GG3-2G4 21227 26174  23 701  GG3-1G915816 18720  17 268  GG3-2G2 11445 10988  11 217  GG3-1D8 10 631  9 85410 243  GG3-5G11  6251  7283 6 767 GG3-1F3  5 553 6 343 5 948 GG3-2E7 6782  8059 7 421 GG3-4F3  6060  5568 5 814 GG3-1F7  3 340 5 158 4 249GG3-2B8  3898  5048 4 473 GG3-4B9  1785  2933 2 359 IF2-2E9 33267 42379 37 823  IF2-2D8 28917 32700  30 809  IF2-IC7 15770 18316  17 043 IF2-ID5 11990 13224  12 607  IF2-2F7  8386  8024 8 205 IF2-4B7  4688 5222 4 955 IF2-3D11  3147  6179 4 663 IF2-3B6 <1600   975   975IF2-2E10 <1600  1234 1 234 IF2-2C4 <1600   761   761

5 clones were kept for each cloid. The 5 clones produced from cloid EE9(EE9-5G7,EE9-5D8, EE9-2F8, EE9-4B1 and EE8-2G10) on average secrete morethan 4.5 μg/mL monoclonal antibodies, the 5 clones from the cloid GG3(GG3-2G4, GG3-1G9, GG3-2G2, GG3-1D8 and GG3-5G11) on average secretemore than 6.5 μg/mL monoclonal antibody and the 5 clones from the cloidIF2 (IF2-2E9, IF2-2D8, IF2-1C7, IF2-1D5 and IF2-2F7) secrete more than 8μg/mL monoclonal antibody.

A cell viability test was conducted for each of these 15 cloids beforefreezing (Table 7) and they were screened for the possible presence ofmycoplasma (FIG. 9).

All the clones had a satisfactory survival rate (>70%) and no clone wascontaminated by mycoplasma.

TABLE 7 viability of the clones selected Viability Viability ViabilityClone (%) Clone (%) Clone (%) GG3-2G4 78 IF2-2F7 79 EE9-2F8 80 GG3-2G283 IF2-1C7 75 EE9-4B1 84 GG3-1D8 86 IF2-2D8 90 EE9-5D8 85 GG3-1G9 89IF2-2E9 71 EE9-2G10 90 GG3-5G11 78 IF2-1D5 95 EE9-5G7 93

Finally 8 clones were selected, based on the following criteria:

-   -   their production of monoclonal antibodies was ≧14 μg/mL    -   their viability was ≧70%

The clones selected were thus the following: EE9-5G7, EE9-5D8, EE9-2G10,GG3-2G4, GG3-1G9, IF2-2E9, IF2-2D8 and IF2-1C7.

Stability of the Clones

The aim of these experiments is to verify that the various clonessecrete substantially the same quantity of monoclonal antibody throughthe different stages.

The eight clones selected were kept for 8 weeks by cascade dilution in24 well plates and subcultured every 7 days. The clones from cloid EE9were kept in a EMS+5% dialysed SVF medium and the clones from cloids GG3and IF2 in a DMEM=5% dialysed SVF medium.

The production of monoclonal antibodies was evaluated in two independentELISA assays and the results are shown in Table 8 below:

TABLE 8 antibody production over time. Name of the Number of AverageAverage Name of the Number of Average Average clones weeks ng/mL μg/mLclones weeks ng/mL μg/mL EE9-2G10 1 29592 30 EE9-5D8 1 0 0 2 4577 5 217072 17 3 9019 9 3 21419 21 4 20163 20 4 18034 18 5 10908 11 5 13317 136 10965 11 6 15010 15 7 7848 8 7 14607 15 8 5191 5 8 8543 9 EE9-5G7 131003 31 GG3-1G9 1 22030 22 2 17087 17 2 18223 18 3 21407 21 3 20848 214 15508 16 4 26141 26 5 12454 12 5 29579 30 6 14053 14 6 31577 32 713466 13 7 39374 39 8 12404 12 8 60901 61 GG3-2G4 1 23966 24 IF2-1C7 118266 18 2 22002 22 2 16667 17 3 23668 24 3 18738 19 4 23469 23 4 1016510 5 32319 32 5 7717 8 6 32727 33 6 6852 7 7 41491 41 7 8158 8 8 4764848 8 8675 9 IF2-2D8 1 39692 40 IF2-2E9 1 29219 29 2 41974 42 2 30861 313 33515 34 3 33623 34 4 30602 31 4 28549 29 5 29631 30 5 27918 28 631373 31 6 36222 36 7 35701 36 7 34909 35 8 35279 35 8 38246 38

The stability of the cloids over time is also represented in graphicform in FIG. 10.

In the end, 3 clones were selected for their stability of monoclonalantibody production: clone EE9-5G7, clone IF2-2D8 and clone IF2-2E9.

Example 3 Production of Antibodies in Roller Bottles According to theInvention

The EE9-5G7 clone was selected for the production of 500 mg ofantibodies on the basis of the dosages of maximum static pseudoproduction which established its level of production as 19 mg/L and alsobased on the results of the stability study conducted on the parentalEE9 cloid, whose production level was stable for six weeks.

On the basis of these data, clone EE9-5G7 was amplified in such a way asto achieve two successive productions of 30 roller bottles in batchmode. Production was stopped when the cell viability dropped below 50%.As the supernatant measured at the end of production was only 12 mg/L,namely 648 mg antibody produced. This supernatant was measured,centrifuged, filtered before being concentrated 15 times. Theconcentrate was purified by affinity chromatography and this allowed 468mg purified antibody to be obtained.

The production of anti-ricin monoclonal antibody by clones IF2-2D8 andIF2-2E9 was achieved by roller bottle production in an RPMI and EMSmedium.

The cells were unfrozen and seeded to 2.10⁵ cells/ml in a DMEM, 5% SVFmedium depleted in Ig, 0.5 g/l geneticine. The viability from unfreezingwas 91% for clone IF2-2D8 and 93% for clone IF2-2E9.

The progress of culture and roller production is shown in Table 9 forclone IF2-2D8 and in Table 10 for clone IF2-2E9.

TABLE 9 Follow-up of the culture and roller production of clone IF2-2D8Subculture Volume of Concentration Viability density Type of medium Date(×10⁶/ml) (%) (×10E6/ml) container (ml) Flask number Medium J0 Change ofmedium T75 30 2 DMEM J3 −10 ml +10 ml medium T75 30 2 DMEM J7 0.64 860.1 T75 30 1 DMEM J10 1.1 95 0.1 T75 30 1 DMEM J14 1.38 96 0.1 T75 30 1DMEM T75 30 1 RPMI T75 30 1 EMS J17 0.9 100 0.2 T175 100 1 RPMI 0.88 980.2 T175 100 1 EMS J20 1.48 97 0.3 Roller 500 1 EMS 1.12 100 0.3 Roller400 1 RPMI J22 0.8 95 0.44 Roller 900 Production EMS Start 0.74 95 0.32Roller 900 Production RPMI Start J24 2.12 93 Roller 900 EMS 0.92 96Roller 900 RPMI J27 Viability <50% Stop Roller EMS Viability <50% StopRoller RPMI

TABLE 10 Progress of culture and roller production of clone IF2-2E9 CellConcentration Viability density Type of Volume of Date (×10⁶/ml) (%)(×1E6/ml) container medium (ml) Flask Number Medium J0 0.56 82 0.1 T7530 1 DMEM J3 0.76 95 0.1 T75 30 1 DMEM J7 1.4 91 0.1 T75 30 1 DMEM 1 EMS1 RPMI J10 0.68 100 0.1 T75 30 1 RPMI 0.9 100 0.1 T75 30 1 EMS J14 0.9898 0.2 T175 100 1 RPMI 1.2 91 0.2 T175 100 1 EMS J17 1.26 95 0.25 Roller500 1 RPMI 1.12 96 0.22 Roller 500 1 EMS J20 1.74 92 0.9 Roller 900Production Start RPMI 1.84 98 0.9 Roller 900 Production Start EMS J221.54 90 Roller 900 1 RPMI 2.1 88 Roller 900 1 EMS J24 Viability <50%Stop roller RPMI Viability <50% Stop roller EMS

The supernatants for these rollers were quantified by ELISA (Table 11),in order to measure the quantity of antibody produced.

TABLE 11 Doses of roller supernatants. Concentration Clones Medium(μg/ml) IF2-2D8 EMS 39 IF2-2D8 RPMI 32 IF2-2E9 EMS 47 IF2-2E9 RPMI 34

The supernatants were filtered through a 0.22 μm filter and theantibodies were purified by affinity chromatography on a HiTrap proteinA FF column.

Purification assessment of the anti-ricin antibodies IF2-2D8 and IF2-2E9

Culture Antibodies supernatant purified Name of Culture IgG Vol. IgGYield clone medium Vol. (ml) (mg) (ml) (mg) (%) IF2-2D8 EMS 1094 27.757.2 21.1 76.2 IF2-2D8 RPMI 1096 21.7 11.6 16.1 74.2 IF2-2E9 EMS 1095 3817.2 32.8 86.3 IF2-2E9 RPMI 1095 24.3 24.3 22 90.5

The concentrates allowed between 16 and 32.8 mg purified antibody to beobtained for each of the clones.

Clones IF2-2D8 and IF2-2E9 proved to be stable during the differentstages (unlike their parental cloid) produce [sic] twice as manymonoclonal antibodies as clone EE9-5G7. The physico-chemicalcharacterisation of the antibodies showed that it is clone IF2-2E9 whichproduced the antibody most like the antibody produced by clone EE9-5G7.For this reason, it was clone IF2-2E9 which was chosen for the nextproductions of anti-ricin antibodies

Production and purification of a batch of 500 mg anti-ricin antibody.

-   -   The culture supernatant for clone IF2-2E9 was concentrated,        filtered, then the antibodies were purified in a first stage by        affinity chromatography on Sepharose protein A followed by two        stages of ion exchange. The quantity of antibody purified is 642        mg.

Example 4 In Vitro Test of Ricin Neutralisation by the Human/MacaqueChimeric Monoclonal Antibody—Survival Test

This test measures the capacity of the antibodies forming the subject ofthe invention to protect J774A.1 cells which have come into contact withRicin from dying.

Briefly, J774A.1 cells (ATCC-LGC, Molsheim, France) were seeded at adensity of 14.000 cells/well (200 μl/well), in a culture plate andcultivated at 37° C. in the presence of 5% CO₂ for 24 hours in a DMEMmedium complemented by 10% fcetal vole serum. The antibodies forming thesubject of the invention were incubated with 480 ng/ml ricin or withcontrol serum (antibodies not relevant) for one hour. The mixture wasthen added to the cells. 24 hours afterwards, cell viability wasmeasured by techniques known to the expert in the field (Trypan BlueExclusion Test, Cytox (Promega), measurement of apoptosis . . . ). Eachtest was corrected in relation to the “100% cell viability” controls (noricin and no antibodies) and “0%” viability (ricin without antibodies).

Example 5 In Vitro Test of Ricin Neutralisation by the Human/MacaqueChimeric Monoclonal Antibody—Translation Inhibition Test

Another test may be used for measuring the neutralising activity of themonoclonal antibodies forming the subject of the invention, this meansmeasuring protein translation inhibition. To do this, the translation ofa marker protein is measured, for example the translation of luciferase,in an acellular in vitro translation system [Hale M L Pharmacol Toxicol2001, 88(5):255-260]. Briefly, the test for measuring the translation ofthe luciferase messenger RNA is the following: The monoclonal antibodiesare placed in 96 well plates in phosphate buffered saline (PBS) andricin is added at a final concentration of 4 mM. Rabbit reticulocytelysate complemented by RNAsin®, amino acids of the luciferase messengerRNA is then added to each well. The reaction is achieved over 1.5 hours.

5 μL of the reaction are then taken and added to 45 μL of rectionalbuffer solution, allowing luciferase activity to be detected (Luciferaseassay reagent, Promega, Inc.). The light emitted (luminescence) ismeasured in counts per second (CPS) with the aid of a Victor multi-platereader (PerkinElmer Wallac, Boston Mass.). The data are expressed as apercentage compared to a control (% control ═(CPS treated/CPScontrol)×100)).

In the two tests (Example 4 and Example 5), the comparison between the50% protecting dose of scFv (1 μg/ml) and that of the IgG (0.5 μg/ml)shows a net benefit of the expression in the form of IgG.

Example 6 In Vivo Test of Ricin Neutralisation by the Human/MacaqueChimeric Monoclonal Antibody

In order to test the neutralising effect of the anti-ricin antibody invivo, Fisher rats (Charles River Laboratories L′Arbresle, France)received a dose of 16 μg/kg by weight of ricin and 50 μg anti-ricinantibody, following an adjustment of the protocol described by Ezzell etal. (Ezzell, et al. 1984. Infect. Immun 45:761-767)

The tests were carried out by measuring the viability of the animals asa function of the doses of ricin and of anti-ricin antibodies injected.

As a control, the viability of the rats treated solely with the ricin(positive test) and the rats treated solely with the anti-ricin antibody(negative test) was also evaluated.

Example 7 Affinity Assay of the Human/Macaque Chimeric MonoclonalAntibody for Ricin

The affinity assay of the anti-ricin antibody for its antigen, that isto say ricin, was measured by surface plasmon resonance by means of aBIAcore X instrument (Biacore, Uppsala, Sweden). The ricin wasimmobilised on a CMS chip (Biacore), by means of an amine coupling, inaccordance with the manufacturer's instructions.

For each measurement, a minimum of six dilutions, in an HBS-EP (Biacore)buffer of anti-ricin antibody was tested for 900s (dilutions from 10 to0.1 μg/mL). After each dilution of the antibody, the chip wasregenerated with 1.5 glycin (Biacore) at a flow of 10 μl/min for 30 s.The affinity constants were calculating by using the method described byKarlsson et al. (Karlsson et al. 1991, J. Immunol. Methods 145:229-240)and checked using internal tests as described by Schuck, et al. (Schuck,P., and A. P. Minton. 1996. Anal. Biochem. 240:262-272).

Example 8 Administration of the Human/Macaque Chimeric MonoclonalAntibody to Mice by Instillation and Determination of Survival

BALb/c mice (20-22 g) were exposed to ricin by pulmonary instillation atthe level of 16 μg/kg body weight and survival was checked 10 days afteradministration of the ricin.

The anti-chain A IgG 43RCA type of ricin antibody or a human IgG controlwere administered by pulmonary instillation at a level of 50 μg. Theprotective effect of the anti-chain A IgG 43RCA type of ricin antibodywas measured by the percentage of mice which survived and by the ratioof wet weight over dry weight in the lungs, taking account of the factthat ricin induces pulmonary oedema.

The mice exposed to the ricin who had received the human control IgGwere all dead by Day 3 3 (0% survival) (n=4). 100% survival was observedin the mice which had received the ricin and the 43RCA antibody in thesame injection (n=2, not represented in FIG. 11)

The mice which had received the 43RCA antibody 6 hours afteradministration of the ricin also revealed 100% survival (n=7)

Among the mice which had received the 43RCA antibody 22 hours after theadministration of the ricin (n=7), 2 died on day 4, 1 died on Day 8 andthe other 4 mice survived. The survival percentage is equal to 57.1%when the antibody is administered 22 hours after the administration ofthe ricin by the pulmonary route. Among the mice which had received the43RCA antibody 24 hours after the administration of the ricin (n=11), 2died on day 4, 2 died on Day 6. On Day 10, 7 of the 11 mice hadsurvived. The survival rate is equal to 63.6%

The ratio of wet weight to dry weight of the lungs was measured 3 daysafter administration of the ricin to the mice, whether or not theyreceived the 43RCA antibody. As shown in the table below, the ratio goesdown if the mice receive the ricin pre-mixed with the 43RCA antibody,demonstrating that this 43RCA antibody neutralises the toxicity of thetoxin and limits oedema of the lungs.

Ratio wet weight/dry weight of the lungs SD Saline Solution 4.47 0.31Ricin 6.56 0.655 Ricin + 43RCA IgG 5.36 0.607

Example 9 Administration of the Human/Macaque Chimeric MonoclonalAntibody to Mice by Instillation and Determination of Minimum Doses

BALb/c mice were exposed to ricin by pulmonary instillation at a levelof 16 μg/kg body weight and received an administration of 43RCA 6 hoursafterwards, as 1, 5, 10, 20 μg.

FIG. 12 shows that whatever the dose administered, 100% survival rate isobserved in the mice. The 43RCA antibody proved to be therapeuticallyeffective from 1 μg upwards when it is administered 6 hours afterintoxication by ricin.

BALb/c mice were exposed to ricin by pulmonary instillation at a levelof 16 μg/kg body weight and survival was checked for 10 days afteradministration of the ricin. The anti-chain A IgG 43RCA type of ricinantibody or a human IgG control were administered by pulmonaryinstillation at a level of 150 mg.

As shown in FIG. 13, the mice which had received the ricin and an IgGcontrol all died on Day 4. The mice which had received the 43RCAantibody 44 hours after the pulmonary administration of ricin had asurvival rate of 83% (n=12). The mice which had received the 43RCAantibody 54 hours after the pulmonary administration of ricin had asurvival rate of 75% (n=8). The 43RCA antibody therefore has atherapeutic effect even when it is administered more than two days afterintoxication by ricin.

Example 10 Administration of the Human/Macaque Chimeric MonoclonalAntibody by Aerosol

The mice were exposed to aerosols containing ricin and the anti-ricinantibody obtained by a Collison nebuliser (mass median aerodynamicdiameter [MMAD]=1.2 urn).

The concentrations of ricin and antibody were identical for eachexposure (ricin=1 mg/ml, antibody=14 mg/ml); the aerosol concentrationbeing determined by the exposure time.

The aerosols were continually measured during exposure by means of aglass impact tester (AGI). The concentration of protein collected in theimpact tester was measured by using the BCA micro protein assay kit(Pierce Co., Rockford, Ill., U.S.A.)

The diffusion conditions for the aerosols and their recovery remainedconstant throughout the study. The aerosol concentration (μg/L) wascalculated and the dose of ricin inhale [sic] (μg/mouse) was estimatedby using the Guyton formula.

1. A chimeric anti-ricin monoclonal antibody, wherein: the constantregion of the light chain essentially comprises the constant region ofthe light chain of a human immunoglobulin; the constant region of theheavy chain essentially comprises the constant region of the heavy chainof a human immunoglobulin; the variable region of the light chaincomprises the variable region of the light chain of a macaqueimmunoglobulin; the variable region of the heavy chain comprises thevariable region of the heavy chain of a macaque immunoglobulin; and themonoclonal antibody does not substantially induce an immune responseagainst chimeric antibodies.
 2. The antibody according to claim 1,wherein the antibody is capable of neutralizing ricin.
 3. The antibodyaccording to claim 1, wherein the antibody is capable of neutralizingricin and has a ricin neutralization rate that is higher than theneutralization rate of a fragment of scFv targeted at ricin.
 4. Theantibody according to claim 1, wherein: the constant region of the lightchain comprises the constant region of the light chain of a kappa typehuman immunoglobulin; and the constant region of the heavy chaincomprises the constant region of the heavy chain of a type IgG1 humanimmunoglobulin.
 5. The antibody according to claim 4, wherein theconstant region of the light chain and the constant region of the heavychain are obtained by immunization with a Rhesus D antigen.
 6. Theantibody according to claim 4, wherein the constant region of the lightchain and the constant region of the heavy chain are obtained from theT125-A2 clone.
 7. A Fab or F(ab)′2 fragment of the monoclonal antibodyaccording to claim
 1. 8. Nucleic acid coding for the light chain of themonoclonal antibody according to claim
 1. 9. The nucleic acid accordingto claim 8, comprising SEQ ID NO: 1, 5, 9 or
 13. 10. Nucleic acid codingfor the heavy chain of the monoclonal antibody according to claim
 1. 11.The nucleic acid according to claim 8, comprising SEQ ID NO: 3, 7, 11 or15.
 12. Nucleic acid coding for the light chain and coding for the heavychain of the monoclonal antibody according to claim
 1. 13. An expressionvector, comprising nucleic acid coding for the light chain, coding forthe heavy chain, or coding for the light chain and the heavy chain ofthe monoclonal antibody according to claim 1, said nucleic acid beingunder control of elements that permit expression of the nucleic acid.14. A pharmaceutical composition, comprising at least one of: themonoclonal antibody according to claim 1; a Fab or F(ab)′2 fragment ofsaid monoclonal antibody; a nucleic acid coding for the light chain ofsaid monoclonal antibody, coding for the heavy chain of said monoclonalantibody, or coding for the light chain and the heavy chain of saidmonoclonal antibody; or a vector comprising said nucleic acid, inassociation with a pharmaceutically acceptable vehicle.
 15. Apharmaceutical composition, comprising at least one monoclonal antibodyaccording to claim 1, in association with a pharmaceutically acceptablevehicle.
 16. A method for treating or preventing a pathology associatedwith ricin contamination in a subject, comprising administering to thesubject at least one of: the monoclonal antibody according to claim 1; aFab or F(ab)′2 fragment of said monoclonal antibody; a nucleic acidcoding for the light chain of said monoclonal antibody, coding for theheavy chain of said monoclonal antibody, or coding for the light chainand the heavy chain of said monoclonal antibody; a vector comprisingsaid nucleic acid; or a cell comprising said monoclonal antibody, saidfragment, said nucleic acid, or said vector.
 17. A method of measuringricin in vitro, in a biological sample from an individual who may havebeen contaminated by ricin, the method comprising: bringing the sampleinto contact with at least one monoclonal antibody according to claim 1;and determining the presence or absence of ricin in said sample bydetecting the formation of an immune complex between the ricin and saidmonoclonal antibody.
 18. A process for decontaminating in vitro, asample that may have been contaminated by ricin, the process comprising:bringing the sample into contact with at least one monoclonal antibodyaccording to claim 1, and removing from said sample any immune complexesformed between the ricin and said monoclonal antibody.
 19. A process forpreparing the antibody according to claim 1, comprising: transforming acell with a vector comprising a nucleic acid coding for the light chainof said monoclonal antibody, coding for the heavy chain of saidmonoclonal antibody, or coding for the light chain of said monoclonalantibody and the heavy chain of said monoclonal antibody; selecting thetransformed cells; and assessing the production of said antibody fromthe selected cells, by determining the presence or absence of theformation of an immune complex between ricin and said monoclonalantibody.
 20. The process according to claim 19, wherein the cell is ofthe cell line YB2/0.